September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
The Role of Notch Signaling in the Regenerating Adult Zebrafish Retina
Author Affiliations & Notes
  • Joshua Hobgood
    Biology , University of Notre Dame, South Bend, Indiana, United States
  • Footnotes
    Commercial Relationships   Joshua Hobgood, None
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Investigative Ophthalmology & Visual Science September 2016, Vol.57, 4189. doi:
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      Joshua Hobgood; The Role of Notch Signaling in the Regenerating Adult Zebrafish Retina. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4189.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : In the damaged zebrafish retina, Müller glia are responsible for regenerating lost cells. We previously showed that Notch signaling is required to maintain Müller glia in a quiescent state, and inhibiting Notch signaling, via intraperitoneal injection of the g-secretase inhibitor RO4929097, is sufficient to induce a regenerative response. Thus, it appears Notch signaling is a negative regulator of retinal regeneration in the zebrafish eye. However, the zebrafish genome encodes four unique Notch receptors; Notch 1a, Notch 1b, Notch 2, and Notch 3. It was unclear if all or only a subset are involved in Müller glia quiescence and if they had any other functions in retinal regeneration. Thus, the purpose of this study was to begin to elucidate the function(s) of the individual Notch receptors in the undamaged and regenerating zebrafish retinas.

Methods : Adult albino zebrafish were placed in complete darkness for 14 days, before being subjected to constant light damage. Retinas were isolated for RNA extraction, and qRT-PCR was performed on the four Notch receptor genes using Taqman probes. Dark-adapted albino zebrafish were subjected to morpholino-mediated knockdown of all four Notch receptors, separately, and then subjected to light damage. The retinas were isolated, cryosectioned, and immunolabeled for PCNA, Stat3, and Ascl1a, and analyzed by confocal microscopy

Results : The qRT-PCR analysis revealed an increase in expression for notch 1a, 1b, and 2, while notch 3 decreased in expression from at from 0 to 16 hours, and increased subsequently throughout the light timecourse. Morpholino-mediated knockdowns of Notch 1a, 1b, and 2 resulted in fewer proliferating Müller glia and neuronal progenitors at 36 and 72hr of light compared to the controls. In contrast, morpholino-mediated knockdown of Notch 3 resulted in increased numbers of proliferating Müller glia at 36 and 72 hrs.

Conclusions : Notch receptors 1a, 1b, and 2, are required for the maximal proliferative response in the light-damaged zebrafish retina. In contrast, downregulation of Notch 3 is necessary for Müller glia to re-enter the cell cycle in response to damage. This suggests that Notch 3 is necessary to maintain Müller glia quiescence and Notch 1a, 1b, and 2 are required for Müller glia and neuronal progenitor proliferation.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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